RNA from T-cells
Anton Scott Goustin
asg at cmb.biosci.wayne.edu
Wed Aug 16 15:25:40 EST 1995
vblasch at gwdg.de (Volker Blaschke) wrote:
>Dear fellow researchers,
>I need to make RNA from Buffy-coat (native) T-cells. However, with all
the protocolls I've used so far (e.g. GuSCN/Ultracentrifugation,
Chomczynski/Sacchi and Qiagen RNeasy kit), yield was incredibly low
(around 5 ug total RNA from 20 million cells).
>Does anyone know if this yield is in fact what one gets from native
T-cells or of another methods giving higher yields?
Qiagen states in their protocoll that their yield is around 5 ug total
RNA from 1 million T-cells, but what they don't say (and can't say on
the phone) is if that's native cells or stimulated cells or a cell line.
You hit on a main point: stimulated versus resting. Resting T cells
have only a thin layer of cytoplasm around their big nuclei. The low
yield of RNA is thus a natural consequence of the resting state. I
normally use direct capture of poly(A+) RNA as a first approach to any
cell or tissue, a method first used by Manfred Schwab in 1982 as a
postdoc in Mike Bishop's lab in San Francisco. My experience here is
dismal: I got almost nothing! It turns out that not only do resting T
cells have little RNA, they have low ratios of poly(A+) RNA to total RNA
and shorter poly(A) tails. The solution is to use hellish numbers of
cells...and, dare I say: RT/PCR! Northerns are an uphill battle. Can
you stimulate your T cells with PHA, cytokines? That will help, if
stimulated is OK. But, you probably have the same problem as I do: you
want to show that the native cells also express the mRNA....
If you are working with humans, as I have, you can always scale up so
several hundred mL of blood (~1 million T cells per mL). Try
That's where my experience ends!
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